PROJECT SUMMARY Substance use disorder (SUD), a chronic, relapsing disorder with limited treatment options, costs the US ~$700 billion annually. A few, moderately effective replacement therapies exist for opiate, nicotine and alcohol dependence. However, no such options exist for stimulant dependence and, further, there are no pharmaco- therapies to prevent or even reduce relapse associated with any drug class. Associations that have formed during periods of drug use serve as powerful relapse factors by triggering motivation to seek the drug. Recently, the synaptic actin cytoskeleton, a critical regulator of memory, was identified as a biological target for the selective disruption of these associations. Indeed, intra-CNS actin depolymerization produces an immediate and persistent loss of well-established memories associated with the stimulant methamphetamine (METH), without affecting other types of memories. However, ?-actin, the isoform implicated in neuronal plasticity, serves critical functions throughout the body. Therefore, efforts turned to an upstream regulator of the actin cytoskeleton, nonmuscle myosin II (NMII), a more selectively expressed molecular motor that promotes synaptic actin polymerization. All results with direct actin depolymerization have been recapitulated with Blebbistatin, a small molecule inhibitor of NMIIs. Importantly, a single treatment with Blebbistatin is sufficient to produce a seemingly permanent loss of METH-associated memories. In addition to its efficacy in animal relapse models, several properties make Blebbistatin an excellent scaffold for medicinal chemistry. Paramount among these are the molecule?s small size, selectivity for myosin IIs, high brain penetration, and rapid clearance from plasma and brain (short-acting is sufficient and reduces unwanted peripheral and central effects). Unfortunately, Blebbistatin?s activity at cardiac muscle myosin IIs is likely a liability. Optimization efforts have been limited because the therapeutic potentials of NMII inhibition have only been realized in the past few years. Thus, the central goal of this proposal is to develop a safe and selective NMII small molecule inhibitor with improved potency and solubility, that retains its existing, advantageous DMPK properties with minimized toxicological parameters. To accomplish the overall goal of developing an IND ready compound, an extensive pharmaceutical approach to drug discovery and development centered on Blebbistatin analogs will be employed, centered on a detailed structure activity relationship approach. A team has been assembled with many years of preclinical drug development experience that encompasses all of the methodologies employed in this project (medicinal chemistry, biochemistry, cell biology, neurobiology, in vivo pharmacology, drug metabolism and pharmacokinetics (DMPK), toxicology, and clinical research), making this team uniquely qualified to direct the program. By capitalizing on the unique therapeutic discovery and development capabilities at Scripps Florida, this proposal is expected to yield a safe, efficacious compound with sufficient preclinical safety data to support human clinical trials focused on SUD relapse.